We propose to elucidate the mechanism of Cas9/CRISPR- (CRISPR associated 9/clustered regularly interspaced short palindromic repeats) initiated gene modification. Specifically, we propose to identify the cellular recombination mechanisms that are engaged when cleavase, nickase or dual nickase versions of Cas9- are utilized and to also begin to identify and characterize the genes that regulate these processes. In the previous funding period we were able to demonstrate (unexpectedly) that rAAV (recombinant adeno- associated virus), a naturally single-stranded virus, uses a double-stranded form (probably a replicative intermediate) to facilitate gene modification. In addition, w unequivocally demonstrated that rAAV-mediated gene targeting events and random insertions are mechanistically distinct and are carried out by separate DNA DSB (double-strand break) repair pathways. In addition, we demonstrated that the introduction of a chromosomal DSB radically altered the way that rAAV carries out gene modification. Lastly, we have identified that the mismatch repair status of a target cell is the single most important factor that determines the susceptibility of that cell to gene-editing activities. In this grant application, we propose to exend our mechanistic studies to Cas9/CRISPR-initiated gene modification. We will do this by defining the cellular recombination pathways that are engaged when cleavase, nickase or dual nickase versions of Cas9 are utilized. Secondly, we will identify and characterize druggable candidate genes that regulate the process of gene modification. Finally, we propose to carry out whole genome screens to identify new factors that regulate gene modification in human somatic cells. The importance of understanding these processes for gene therapy is clear. Thus, the ability to perform safe, high-frequency molecular surgery on human cells is paramount to realizing the goal of gene therapy. In a larger view, we are investigating pathways of DNA DSB repair that - besides gene modification - regulate many other cellular processes including genomic stability. Thus, the likelihood of discovering ancillary insights into immortalization, aging, tumorigenesis etc. lends confidence to the belief that these proposed experiments are important. To our knowledge, we are one of only a few laboratories in the world utilizing genetic, loss-of-function approaches to study gene modification in human cells and thus we are well-positioned to gain insight into the mechanism of gene editing that cannot be obtained elsewhere.